Endotoxin is lipopolysaccharide (LPS) present in outer membrane of cell wall of gram-negative bacteria, and has been known as a potent pyrogen. For this reason, detection of endotoxin is considered important in the injectable pharmaceuticals and the like, and the test method of endotoxin has been described in pharmacopeia of the United States, Japan and other countries. In addition, the endotoxin is considered to be a main cause of a shock in gram-negative bacterial infection, and in the clinical diagnosis, determination of plasma endotoxin level is used for the diagnosis of gram-negative bacterial infection, determination of therapeutic effect and prognosis of therapy for gram-negative bacterial infection, and earlier diagnosis for endotoxin shock and so on. On the other hand, β-glucan is known as one of the main cell wall constituents of many pathogenic fungi, and in the clinical diagnosis, determination of plasma or serum β-glucan level is employed for earlier diagnosis of the fungal infection, and determination of therapeutic effect and prognosis.
With respect to the method for determining endotoxin and/or β-glucan, a variety of methods through the utilization of effects of plural protease precursors (Factor C, Factor G, Factor B, proclotting enzyme, coagulogen) which are activated by the reaction of an amebocyte lysate of a horseshoe crab with endotoxin and/or β-glucan, among them, methods utilizing chromophore or fluorescent group which greatly changes intensity of color or fluorescence by cleaving amide bond lying adjacent to the chromophore or the fluorescent group, namely, methods for determination using synthetic peptide derivatives, in which a chromophore compound or a fluorescent compound such as nitrophenol, nitroaniline, coumarin derivative and indoxyl derivative is introduced through amide bond, as a substrate for endotoxin measurement have been reported (Patent Literature 1, Patent Literature 2, Patent Literature 3, Patent Literature 4). However, in each case of the determination methods employing such substrates, a chromophore or a fluorescent group which changes intensity of color or fluorescence by cleaving amide bond lying just adjacent to the chromophore group or the fluorescent group has been introduced. As a result, these methods had a problem of not applicable to highly sensitive determination due to the reasons such as low sensitivity of determination, and influence of interfering substances in the serum being unavoidable because excitation wavelength of the released substance was in a range of that possessed by the interfering substances.
Patent Literature 1: JP-B-59-19532
Patent Literature 2: JP-B-61-54400
Patent Literature 3: JP-A-57-502266
Patent Literature 4: JP-A-8-34796